Fatty acids are important components of lipids such as phospholipids and triacylglycerols. Fatty acids containing two or more unsaturated bonds are collectively referred to as polyunsaturated fatty acids (PUFA) and are known to include arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. Various physiological activities have been reported for these fatty acids (Non-patent Document 1). These polyunsaturated fatty acids are expected to have applications in various fields, but some of them cannot be synthesized in the animal body. Thus, microbial techniques have been developed for obtaining polyunsaturated fatty acids by culturing various microorganisms. Other attempts have also been made to produce polyunsaturated fatty acids in plants. In these cases, polyunsaturated fatty acids are known to be accumulated, for example, as components of storage lipids such as triacylglycerols within microorganism cells or plant seeds.
More specifically, triacylglycerols are produced in vivo as follows. Namely, glycerol-3-phosphate is acylated by glycerol-3-phosphate acyltransferase to form lysophosphatidic acid, which is then acylated further by lysophosphatidic acid acyltransferase to form phosphatidic acid. This phosphatidic acid is, in turn, dephosphorylated by phosphatidic acid phosphatase to form diacylglycerol, which is then acylated by diacylglycerol acyltransferase to form triacylglycerol. Other enzymes such as acylCoA:cholesterol acyltransferase and lysophosphatidylcholine acyltransferase are also known to be indirectly involved in biosynthesis of triacylglycerols.
In the above pathway of triacylglycerol biosynthesis or in the pathways of phospholipid biosynthesis, the reaction in which glycerol-3-phosphate is acylated to form lysophosphatidic acid is known to be mediated by glycerol-3-phosphate acyltransferase (hereinafter also referred to as “GPAT”; EC 2.3.1.15).
GPAT genes have been reported so far in several organisms. As mammalian GPAT genes, two types of genes have been cloned: microsomal (membrane-bound) and mitochondrial (membrane-bound) GPAT genes (Non-patent Document 2). Likewise, as plant GPAT genes, three types of genes have been cloned: microsomal (membrane-bound), mitochondrial (membrane-bound) and chloroplast (free) GPAT genes (Non-patent Document 3). In addition, as GPAT genes derived from the fungus Saccharomyces cerevisiae, two types of genes have been cloned: microsomal (membrane-bound) GPT2 (GATT) and SCT1 (GAT2) genes (Non-patent Document 4). For these fungal genes, it has been shown that GPT2 has the ability to use a wide range of fatty acids covering from palmitic acid (16:0) to oleic acid (18:1) as a substrate, whereas SCT1 has a strong selectivity in using a C16 fatty acid (e.g., palmitic acid (16:0), palmitoleic acid (16:1)) as a substrate (Non-patent Document 4). Moreover, GPAT genes have also been cloned from many other organisms.
Reports are also issued for GPATs derived from microorganisms of the genus Mortierella, which are lipid-producing fungi. With respect to GPAT derived from Mortierella ramanniana, microsomal GPAT has been isolated and found to use oleic acid (18:1) as an acyl donor with 5.4-fold selectivity compared to palmitic acid (16:0) (Non-patent Document 5). With respect to GPAT derived from Mortierella alpina (hereinafter also referred to as “M. alpina”), the microsomal fraction of this fungus has been reported to have glycerol-3-phosphate acyltransferase activity (Non-patent Document 6). In addition, when reacted in vitro with various acyl-CoAs, GPAT present in microsomes of M. alpina (which is in a membrane-bound state) has been found to use a wide range of PUFAs including oleic acid (18:1), linolic acid (18:2), dihomo-γ-linolenic acid (DGLA) (20:3) and arachidonic acid (20:4) as a substrate while retaining high activity (Patent Document 1). Moreover, when expressed in Yarrowia lipolytica transformed to allow biosynthesis of up to eicosapentaenoic acid (EPA), GPAT cloned from M. alpina (ATCC #16266) has been found to provide a higher ratio of dihomo-γ-linolenic acid (DGLA) (20:3) and a lower ratio of oleic acid (18:1) among all fatty acids. This result indicates that PUFA with a longer chain length and a higher unsaturation degree is more selectively incorporated (Patent Document 2).    Patent Document 1: International Publication No. WO2004/087902    Patent Document 2: US Patent Publication No. 2006/0094091    Non-patent Document 1: Lipids, 39, 1147 (2004)    Non-patent Document 2: Biochimica et Biophysica Acta, 1348, 17-26, 1997    Non-patent Document 3: Biochimica et Biophysica Acta, 1348, 10-16, 1997    Non-patent Document 4: The Journal of Biological Chemistry, 276 (45), 41710-41716, 2001    Non-patent Document 5: The Biochemical Journal, 355, 315-322, 2001    Non-patent Document 6: Biochemical Society Transactions, 28, 707-709, 2000